The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to elucidate the mechanism of action of the phorbol esters and their endogenous analog, the lipophilic second messenger sn-1,2-diacylglycerol. Protein kinase C (PKC) is the major receptor for these compounds, and our emphasis is correspondingly directed at his family of isozymes. In a collaborative effort with the groups of Victor Marquez, NCI, Alan Kozikowski, Georgetown University, and Shaomeng Wang, University of Michigan, we combine mutational analysis and pharmacological characterization with computer modeling and chemical synthesis to probe ligand - PKC interactions. We have shown that different ligands, such as phorbol 12,13-dibutyrate, indolactam V, thymeleatoxin or ingenol 3-benzoate, use different sets of contacts to achieve high affinity binding. Our emerging insights contribute to ligand design, and we now have synthetic ligands with nanomolar affinity and with unique selectivity. For example, we find that a constrained diacylglycerol analog induced apoptosis only through PKC delta in LNCaP cells, whereas phorbol 12-myristate 13-acetate, induced apoptosis through both PKC alpha and delta. Other studies seek to characterize novel classes of PKC modulators identified from database analysis. We find that the iridals, derived from the iris plant, represent a novel structural motif for binding and possess modest selectivity for the RasGRP family of novel diacylglycerol receptors. Although PKC is the major receptor for the phorbol esters, several novel families of proteins with C1 domains have now been identified which also recognize phorbol esters with high affinity. The RasGRP family members, activators of low molecular weight GTPases such as Ras and Rap, are of particular interest. Phorbol esters bind RasGRP1 and RasGRP3 with similar affinity to PKC, induce translocation in intact cells, and lead to activation of Ras and of Erk1/2, downstream targets of Ras. Localization of RasGRP3 in response to ligands depends on specific concentration. Whereas lower concentrations cause plasma membrane localization, higher concentrations cause localization to the nuclear membrane and perinuclear region. Since localization drives access to its targets, such differential localization may control RasGRP3 specificity. The phorbol related diterpene resiniferatoxin acts as an ultrapotent analog of capsaicin and has permitted characterization of specific capsaicin (vanilloid) receptors. Its differential ability to desensitize vanilloid receptors makes it an attractive therapeutic candidate for treatment of chronic pain and other conditions in which vanilloid sensitive pathways are involved (e.g. urge incontinence). Structure-function studies of the cloned vanilloid receptor, VR1, are beginning to reveal the regions of the receptor involved in ligand recognition. Using strategies developed for constrained diacylglycerols, we and our collaborator (J. Lee, Seoul National University) have designed high affinity agonists orders of magnitude more potent than capsaicin. We likewise have developed multiple classes of competitive antagonists. In addition to capsaicin, heat and protons are activators of VR1. We find that different antagonists can selectively modulate these different activators. On-going work seeks to further characterize the properties of our novel analogs. In related work, we seek to understand the role of signaling pathways, in particular phosphorylation and dephosphorylation, on VR1 function. At the structural level, we have shown by both molecular and biochemical techniques that VR1 exists as a tetramer. The contribution of this oligomeric structure to VR1 function is under investigation.